A New Co-Expressed P450 and UGT Platform for the Simultaneous Preparation of P450 Metabolites and Their Glucuronides

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Toxins, pharmaceuticals and physiologically indogenous compounds are metabolized by the liver via several classes of enzymes. The uridine diphosphate glucuronosyltransferases (UGTs) are concentrated in the liver but also present in other tissues. These enzymes add UDP-glucuronic acid (UDPGA) to a compound, thereby increasing its solubility and ability to be excreted in the bile and urine. Oxford Biomedical Research has created a proprietary method for processing a strain of Pichia pastoris optimized for expressing the cytochromes P450 (CYP) and oxidoreductase, resulting in a growing product line tailored to pharmaceutical research. This process transforms the yeast into a stabilized dry powder (SDP) format, which exhibits remarkable organic solvent and thermal tolerance along with extended catalysis when compared to traditional recombinant enzymes. The proposed multi-catalytic platform, capable of simultaneous P450 oxidation followed by UGT glucuronidation, would give the pharmaceutical researcher a valuable tool for preparing glucuronides as well as studying the P450/UGT synergistic relationship. This Phase I proposal will involve producing and optimizing three catalytic systems:the first Pichia system will express the dehydrogenase responsible for converting UDP-glucose to UDPGA, the cofactor required for glucuronide production. The second system will include expand on this platform and include the UGT2B7 enzyme to create a catalyst capable of glucuronidating 4-methylumbelliferone. The third and final system will add into this expression system the CYP3A4 and P450 oxidoreductase (POR) enzymes, with development focusing on the optimization of 1'-hydroxymidazolam glucuronide production. Achieving these goals will give pharmaceutical research a robust, easy-to-use system for performing two metabolically-important reactions in one system; P450 oxidation followed by UGT glucuronidation.